RESUMEN
The human alveolar macrophage product, enzyme-releasing peptide (ERP), has a molecular mass of 8,000 Da, and releases azurophilic and specific granule constituents from neutrophils. A murine monoclonal anti-ERP antibody (12E10H), previously used to show a lack of antigenic identity between ERP and C5a, interleukin 1, tumor necrosis factor, and gamma-interferon, showed no cross-reactivity with interleukin 8. 12E10H and a fluorescein-labeled second antibody were used to visualize ERP on the macrophage surface. ERP was removed from alveolar macrophages by a 3-min incubation with 5 X 10(-7) M bovine pancreatic trypsin at 37 degrees C. The washed trypsinized cells could readhere to plastic and exclude trypan blue. Dilution of the trypsin-derived ERP released myeloperoxidase from cytochalasin-B-treated neutrophils dose dependently. The enzyme-releasing ability of the trypsin-derived material was removed by immunoprecipitation using antibody 12E10H bound to Staphylococcal protein A Sepharose 4B. The estimated molecular mass of the trypsin-derived ERP (by molecular sieve chromatography on HPLC) was approximately 8,500 Da. Other proteases (plasmin, thrombin, and cathepsin G) also released ERP from the cell surface, but the ERP was not an active secretagogue for neutrophils. However, macrophages cultured with protease inhibitors did not show decreased ERP accumulation in the medium. Our data indicate that ERP exists on the surface of human alveolar macrophages and can be released by proteases found within the lung environment in some disease states.
Asunto(s)
Macrófagos/fisiología , Neutrófilos/enzimología , Péptidos/metabolismo , Anticuerpos Monoclonales , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Células Cultivadas , Cromatografía Líquida de Alta Presión , Reacciones Cruzadas , Citocalasina B/farmacología , Humanos , Immunoblotting , Neoplasias Pulmonares/patología , Peso Molecular , Neutrófilos/efectos de los fármacos , Péptidos/análisis , Péptidos/farmacología , Péptidos/fisiología , TripsinaRESUMEN
Alveolar fibrin deposition commonly occurs in the lungs of patients with the adult respiratory distress syndrome (ARDS). Bronchoalveolar lavage (BAL) from patients with ARDS, control patients with interstitial lung disease (ILD), congestive heart failure, or exposure to hyperoxia, and normal healthy subjects was studied to determine whether local alterations in procoagulant activity favor alveolar fibrin deposition in the lungs in ARDS. Procoagulant activity capable of shortening the recalcification time of plasma deficient in either factor VII or factor VIII was observed in unconcentrated BAL of all patients, but was significantly greater in BAL from patients with ARDS when compared with that of control subjects (p less than 0.001). Unconcentrated BAL from patients with ARDS shortened the recalcification time of plasma deficient in factor X, but no functional thrombin was detectable. BAL procoagulant from patients with ARDS was inhibited by concanavalin A, an inhibitor of tissue factor. The hydrolysis of purified human factor X by BAL from the ARDS and other patient groups was determined by measuring the amidolytic activity of generated factor Xa on its N-benzoyl-L-isoleucyl-L-glutamyl-glycyl-L-arginine-p-nitroanilide substrate. The procoagulant activity of BAL was associated with the development of amidolytic activity, indicating activation of factor X. BAL from patients with ARDS contained more factor X activating activity than did BAL from control groups (p less than 0.001). This activity was calcium dependent and was maximal at 1 mM ionized calcium. The BAL factor X activating activity was most active at neutral pH and was sedimented by ultracentrifugation at 100,000 x g.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Coagulación Sanguínea , Líquido del Lavado Bronquioalveolar/análisis , Factor VII/fisiología , Síndrome de Dificultad Respiratoria/sangre , Tromboplastina/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Pruebas de Coagulación Sanguínea , Líquido del Lavado Bronquioalveolar/citología , Factor VII/análisis , Factor X/análisis , Factor X/fisiología , Insuficiencia Cardíaca/sangre , Humanos , Enfermedades Pulmonares/sangre , Persona de Mediana Edad , Oxígeno/administración & dosificación , Fibrosis Pulmonar/sangre , Sarcoidosis/sangre , Tromboplastina/análisisRESUMEN
A monoclonal antibody was developed against an 8,000-kDa enzyme-releasing peptide (ERP) released from human alveolar macrophages. ERP was isolated on an immunoaffinity column containing the antibody bound to staphylococcal protein A-Sepharose. Release of ERP from the macrophages is not changed by plastic adherence, phagocytosis, calcium ionophore, or phorbol esters. The peptide was not antigenically similar to interferon-gamma, tumor necrosis factor, or interleukin 1 alpha or 1 beta. The release of constituents from azurophilic and specific granules was the main identified biologic function of ERP. ERP was a more effective secretagogue in the untreated neutrophils and f-met-leu-phe was more effective in the cytochalasin B-treated neutrophils. Absorption of ERP from macrophage-conditioned medium removed a small amount of the chemotactic activity; however, the immunopurified peptide was not chemotactic or chemokinetic for neutrophils, and at high concentrations, it suppressed base line chemokinesis. Treatment of washed macrophages with trypsin released active ERP of approximately the same m.w. of spontaneously secreted ERP. These studies showed that human alveolar macrophages release a peptide which is a secretagogue for human neutrophils under conditions which may be encountered in the lungs during certain disease states. Proteolytic enzymes which are free in the lungs may release the peptide and lead to the secretion of neutrophil enzymes.
Asunto(s)
Exocitosis/efectos de los fármacos , Macrófagos/metabolismo , Neutrófilos/efectos de los fármacos , Péptidos/farmacología , Anticuerpos Monoclonales/inmunología , Quimiotaxis/efectos de los fármacos , Citocalasina B/farmacología , Gránulos Citoplasmáticos/metabolismo , Humanos , Neoplasias Pulmonares/patología , Persona de Mediana Edad , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/metabolismo , Péptidos/inmunología , Péptidos/metabolismo , Alveolos Pulmonares/patología , TripsinaRESUMEN
Although pulmonary fibrin deposition and coagulation abnormalities have been observed in acute lung injury in humans, their role in the pathogenesis of pulmonary disorders is unclear. In order to gain further insights into the role of the coagulation in lung injury, we examined the relationship between procoagulant activity in bronchoalveolar lavage (BAL) fluids and the evolution of bleomycin-induced lung injury in marmosets. The BAL procoagulant activity was increased at 1, 2, and 4 wk after bleomycin challenge compared with that in control subjects, and it was capable of shortening the recalcification times of plasmas deficient in factor VII and factor VIII but not in factor X. This profile suggested the presence in BAL of an activator of factor X. Activation of purified human factor X by BAL was demonstrated by measuring the amidolytic activity of the generated factor Xa on its N-benzoyl-L-isoleucyl L-glutamyl-glycyl-L-argenine-p-nitroanilide substrate. Factor X activating activity was increased in BAL at 2 wk after bleomycin challenge. Cleavage of 125I-labeled human factor X by BAL from bleomycin-challenged marmosets yielded a 55,500 Mr product that comigrated with factor Xa, the appearance of which correlated strongly with amidolytic evidence of factor Xa activity. Electron microscopy of the lungs of animals from all groups revealed pulmonary fibrin deposition at 2 wk after bleomycin challenge, at the time of increased BAL procoagulant and factor X activating activity. The BAL procoagulant activity was completely sedimentable by ultracentrifugation and was inhibited by concanavalin A and phospholipase C. Activation of purified factor X by BAL was inhibited by monospecific polyclonal goat and rabbit antibodies to human factor VII as well as antibody to bovine tissue factor, demonstrating that factor X activating activity in BAL was attributable to tissue factor associated with material similar to factors VII or VIIa. We conclude that procoagulant activity in BAL increases after bleomycin challenge in marmosets and is attributable to activation of factor X by tissue factor associated with factors VII or VIIa-like material. Increased BAL procoagulant activity is temporally associated with pulmonary fibrin deposition and pulmonary fibrosis during bleomycin-induced pulmonary injury in the marmoset.